FIGURE 4.

Hydratase and dehydrogenase activity of recombinant AtMFP2 and AtAIM1. Neither AtMFP2 nor AtAIM1 efficiently degrade enoyl chains longer than C14-CoA. A, in situ 2-trans-enoyl-CoA substrate synthesis with a mixture of acyl-CoA oxidases were monitored at A₂₆₀. B, upon full conversion 0.5 nm MFP2 (black), AIM1 (gray), or buffer were added and the hydratase activity determined by recording A₂₆₀ decrease at 27 °C. After the initial determination, additional 5 nm MFP was added to the reactions to secure full conversion. C, after the reactions had run to completion, 1 mm NAD⁺ was added to each reaction, and the dehydrogenase activity was determined by recording the A₃₄₀ increase at 27 °C. D, after 1 h at 27 °C total production of NADH was determined by recording the A₃₄₀. No C16-CoA substrate was fed to this reaction from the hydratase reaction in B. Each reaction consisted of about 50 μm acyl-CoA substrate, AtACX1, AtACX3 and AtACX4 each at 150 nm, 175 mm Tris-HCl, pH 8.5, 2.5% (w/v) polyethylene glycol 400, and 40 pm catalase.